利用细胞毒性实验检测血液中炭疽毒素致死因子的方法学研究

Development of a cytotoxicity-based method for detection of anthrax toxin lethal factor in blood

目的探索利用炭疽毒素致死因子(lethal factor,LF)的细胞毒性检测血液中LF的可行性,为炭疽的临床检测、治疗及相关研究提供支持。方法利用小鼠巨噬细胞系J774A.1对LF细胞毒性的高度敏感性,检测血液中LF的浓度,对实验条件进行优化,并利用Fischer 344大鼠模型验证该方法的可行性。结果细胞毒性实验能够检测到血浆中的LF浓度可低至5 ng/ml,该方法操作简单,灵敏度高,并在大鼠模型中得到了验证,利用本法测定LF在大鼠血液中的半衰期为4.8～6.5 h。结论初步建立了利用细胞毒性实验检测血液中LF的方法,该方法在灵敏性、经济性和实用性等方面具备一定优势,为炭疽相关研究及临床检测提供了新的选择。

Objective To establish a simple,rapid and sensitive method for detection of anthrax lethal factor in blood.Methods J774A.1 Cell,which is highly sensitive to anthrax lethal toxin,was used as an indicator to detect LF in blood.The protective antigen（PA） and blood plasma concentration was optimized to yield higher sensitivity in the assay.Verification was carried out with the samples from Fischer rats injected with anthrax lethal factor.Results The PA and blood plasma concentration was optimized and a cytotoxicity-based method,which could detect as low as 5 ng/ml LF in the plasma,was established.Samples from Fischer rats injected with anthrax lethal factor could be measured accurately and the half life of LF in the blood of rats was estimated at 4.8-6.5 h.Conclusion A cytotoxicity-based method for detection of anthrax toxin lethal factor in the blood plasma is developed,which exhibits advantages in sensitivity,economy,and practice while facilitating clinical detection of anthrax toxins,therapy and basic research of anthrax.